Abstract/Summary Stem cells are responsible for tissue homeostasis and for protective responses to aging or damage. This behavior requires contact with a well-designed niche: one positioned accurately, and with its constituent cells properly organized. It is therefore important to understand how a niche forms. Unfortunately, most niches are either not well defined, or not tractable at the necessary resolution. For this reason, I have chosen a model system to study niche development, the embryonic Drosophila male gonad. In adult testes, the niche is well-defined, and is a paradigm for studying stem cell-niche interactions. Until now, morphogenesis of this niche, which occurs in the embryo, has only been analyzed in fixed preparations. Our lab recently pioneered live-imaging of the entirety of niche development. This approach revealed key cell behaviors during morphogenesis: 1) Polarized assembly of prospective niche cells at the gonad anterior, and 2) Compaction of the niche into a stratified sphere. I propose to uncover signaling and cell mechanics that guide these processes. Aim 1 will define the source and identity of cues directing niche Assembly. My experiments have clearly implicated the visceral mesoderm (Vm) as the tissue guiding niche placement. I will genetically ablate sub-regions of the Vm to determine the portion required to guide niche assembly. I will then use molecular cloning techniques to generate a driver that specifically expresses in the relevant sub-region of the visceral mesoderm. To identify guidance cues, I will use the driver to drive RNAi against transmembrane and secreted factors that are expressed in the Vm. For candidate cues where there is no RNAi line directed against the cue, I will use CRISPR to engineer a GFP-tagged version of the gene, and then employ deGradFP knockdown. Candidates with a phenotype will be verified by misexpressing them and testing for changes in niche position. Aim 2 will identify the forces affecting Compaction and identify the signals that regulate those forces. I will use quantitative cell mechanics assays and post acquisition image analysis. I will utilize laser ablation to disrupt candidate pulling forces directing niche cell ingression, and test changes to ingression frequency. I will disrupt candidate signaling pathways, such as Hedgehog and Notch, and assess changes to niche area and circularity, and to cell bond tension along forming niche-stem cell interfaces. These experiments will define the physical forces and signaling required to form the niche structure required for proper communication with the stem cells.